In E. coli, the synthesis of ribosomes is controlled primarily at the stage of rRNA transcription initiation. rRNA can account for as much as 50% of instantaneous cellular RNA synthesis. In order to understand the biosynthesis of the protein synthetic apparatus, factors involved in the upstream activation and growth rate dependent regulation of rRNA synthesis are being investigated. The mechanism of rRNA promoter-RNA polymerase association is being studied using kinetic and footprinting methods. This will lead to the identification of the individual steps involved in transcription initiation at the rrnB PL promoter and the factors that influence each step. The specific DNA sequences required for upstream activation and growth rate control will be defined by construction and selection of site mutations, followed by assay of operon fusions in vivo. Finally, the importance of position, orientation, and conformation of the upstream activator sequence (UAS) relative to the promoter and the feasibility of using the UAS to enhance transcription from heterologous promoters will also be examined using operon fusions in vivo.